Biologically active conjugate of a biopolymer and therapeutic agent

ABSTRACT

A biologically active conjugate is disclosed comprising a biopolymer and a therapeutic agent joined by a disulfide bond. The conjugate, when formulated in a pharmaceutical composition with a suitable carrier, has improved in vivo stability and activity, and can be targeted to a variety of cells, tissues and organs.

This application is a divisional application of U.S. patent applicationSer. No. 09/784,402 filed on Feb. 15, 2001, now U.S. Pat. No. 6,749,865,which claims priority to U.S. Provisional Application No. 60/182,558filed Feb. 15, 2000 and to U.S. Provisional Application No. 60/211,508filed Jun. 14, 2000.

This invention relates to the chemical modification of biopolymers forthe delivery of therapeutic agents, such as therapeutic proteins, tospecific tissues, organs or cells within a subject, or to extend thebioavailability of the therapeutic agent by enhancing its in vivostability. The biopolymer is initially modified to introduce one or moredisulfide bonds into a side chain of the biopolymer. This facilitatesthe reaction of the biopolymer with a therapeutic agent that has alsobeen modified to present a reactive thiol moiety to form thebiopolymer-therapeutic agent conjugate. The site-specific reaction ofthe biopolymer and the therapeutic agent increases the stability of thetherapeutic agent upon delivery to the desired site targeted by thebiopolymer.

BACKGROUND OF THE INVENTION

Biopolymers are biocompatible polymers that are useful for a widevariety of biomedical applications, such as for surgical aids, toprevent or reduce the formation of surgical adhesions, and for drugdelivery applications. Many biopolymers are naturally occurringsubstances found in the body, and therefore do not have any unacceptabletoxic or injurious effects on biological function. An example of such abiopolymer is hyaluronic acid (“HA”), a naturally occurringmucopolysaccharide found, for example, in synovial fluid, in vitreoushumor, in blood vessel walls and the umbilical cord, and in otherconnective tissues. Hyaluronic acid consists of alternatingN-acetyl-D-glucosamine and D-glucuronic acid residues joined byalternating β 1-3 glucuronidic and β 1-4 glucosaminidic bonds, so thatthe repeating unit is -(1→4)-β-D-GlcA-(1→3)-β-D-GlcNAc-. In water,hyaluronic acid dissolves to form a highly viscous fluid. The molecularweight of hyaluronic acid isolated from natural sources generally fallswithin the range of 5×10⁴ up to 1×10⁷ daltons.

U.S. Pat. No. 4,582,865, to Balazs et al. states, inter alia, thatcross-linked gels of HA can slow the release of a low molecular weightsubstance that is dispersed therein but not covalently attached to thegel macromolecular matrix. See, also, U.S. Pat. No. 4,636,524, whichcontains a disclosure of related technology. Both of these patentsdescribe HA compositions in which the HA is crosslinked by reaction withdivinyl sulfone, and the use of the crosslinked HA compositions in drugdelivery applications.

R. V. Sparer et al., 1983, Chapter 6, pages 107-119, in T. J. Roseman etal., Controlled Release Delivery Systems, Marcel Dekker, Inc., New York,describes sustained release of chloramphenicol covalently attached tohyaluronic acid by an ester linkage, either directly or in an estercomplex including an alanine bridge as an intermediate linking group.The HA is modified by attaching cysteine residues to the HA via amidebonds, and then the cysteine-modified HA is crosslinked by formingdisulfide bonds between the attached cysteine residues. Similarly, I.Danishefsky et al., 1971, in Carbohydrate Res., Vol. 16, pages 199-205,describe the modification of a mucopolysaccharide by converting thecarboxyl groups of the mucopolysaccharide into substituted amides byreacting the mucopolysaccharide with an amino acid ester in the presenceof 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (“EDC”)in aqueous solution. See, also, U.S. Pat. No. 4,937,270 and U.S. Pat.No. 5,760,220 which describe the modification of hyaluronic acid byreaction of the carboxyl groups of the biopolymer with a nucleophile toproduce a water insoluble amide, and the use of those compositions forcontrolled release drug delivery.

A series of patents assigned to VivoRx Pharmaceuticals, Inc., describecompositions for the in vivo delivery of insoluble pharmaceuticallyactive agents. Delivery of the drug substances is achieved, forinstance, by encasing the active agent in a polymeric shell formed froma biocompatible polymer. The biocompatible polymer may be protein,lipid, DNA molecule or polysacharide, and the pharmaceutically activeagent may be a therapeutic protein such as taxol. The polymer containscovalently attached sulfhydryl groups or disulfide linkages which can becrosslinked to form disulfide bonds. The polymeric shell is formed usingultrasonic irradiation techniques. These compositions are described asbeing less toxic, being more soluble, and having improved targeting ascompared to prior art compositions. Relevant VivoRx patents include U.S.Pat. Nos. 5,498,421; 5,439,686; 5,362,478; 5,635,207; 5,560,933;5,635,207 and 5,639,473.

U.S. Pat. No. 5,496,872 relates to biocompatible and biodegradablecrosslinkable polymers having reactive thiol groups. The reactive thiolgroups can be crosslinked to form disulfide linkages between adjacentmolecules, resulting in a three dimensional network. These polymers canbe used for binding tissues or binding tissues with implantedbiomaterials.

U.S. Pat. No. 5,932,552 describes a keratin hydrogel having biomedicalapplications. The hydrogel is formed from crosslinked keratin bound bydisulfide linkages. Among the biomedical applications described in thepatent are uses of the hydrogels for cell scaffolding in tissue repair.

U.S. Pat. Nos. 5,354,853 and 5,451,661 describe, respectively, thepreparation of phospholipid-saccharide conjugates, and lipids conjugatedto biologically active agents such as peptides, proteins and nucleicacids. These conjugates are described as being particularly useful indrug delivery applications.

U.S. Pat. No. 5,902,795, to Toole et al., discloses hyaluronic acidoligosaccharides, having between one and sixteen repeating units, whichare used to treat tumors in mammals. The patent states that theoligosaccharides act to reduce the level of membrane-associatedhyaluronan-binding proteins, which are expressed on the surface ofcertain tumor cells during cell migration. The treatment is believed toreduce the incidence of tumor metastasis in the mammals.

A. Burnkop-Schnurch et al., J. Controlled Release, 2000, 66, 39,describes the synthesis of carboxymethyl cellulose (“CMC”) andpolycarbophil modified with L-cysteine using carbodiimide chemistry. Thepolymers are reacted with the cysteine to form an amide bond between theprimary amino group of the amino acid and the carboxylic acid of thepolymer. The thiolated polymers were allowed to oxidize to formdisulfide bridges. The dissolution of these tablets, both with andwithout drugs, was analyzed. The tablets were found to have improvedstability and viscoelasticity.

Copending U.S. patent application Ser. No. 09/430,857, now abandoned,relates to surfaces that have been modified by the attachment ofhyaluronic acid. The surface can be part of a medical device, such as astent or a surgical tubing. The surface is modified to include areactive amino group that reacts with a derivatized hyaluronic acid. Themodified devices and instruments are hydrophilic, and have anti-foulingand anti-platelet adhesion characteristics, thereby producing areduction in risks associated with thrombosis.

The conjugated biopolymers of this invention represent a significantimprovement over drug delivery vehicles of the prior art due, in part,to the site-specific reaction between the biopolymer and the therapeuticagent which increases the stability and activity of the therapeuticagent upon delivery to the desired site within a subject.

SUMMARY OF THE INVENTION

The present invention features a biopolymer-therapeutic agent conjugatein which the biopolymer and therapeutic agent are joined by a disulfidebond. The biologically active conjugate of this invention is useful as adrug delivery vehicle for the in vivo delivery of the therapeuticproteins to specific cells, organs or tissues in a subject. Drugdelivery specificity is achieved by appropriate selection of thestructure and molecular weight of the biopolymer.

The chemistry used to prepare the conjugates permits the site-specificreaction between the biopolymer and the therapeutic agent. Thetherapeutic agent contains a reactive thiol group, which can be presentin an unmodified version of the therapeutic agent, as in the case ofcysteine for example. Alternatively, the thiol group can be introducedinto a modified version of a therapeutic agent that does not normallycontain a reactive thiol group.

In one embodiment, the therapeutic agent can be reacted, through thereactive thiol group, with a chemically modified version of thebiopolymer. This reaction typically occurs at a pH in the range of fromabout 6.0 to about 10. The biopolymer is activated and modified byreaction with an activating agent, such as a carbodiimide, and reactedwith an organic disulfide compound. The organic disulfide compoundcontains a terminal group, such as an amino group or a hydroxyl group,which is reactive with the carboxylic acid group of the biopolymer inthe presence of the activating agent. The reaction of the biopolymer,activating agent and organic disulfide compound occurs at a pH of fromabout 2.0 to 8.0.

In another embodiment, the therapeutic agent can be reacted, againthrough the thiol group, with the reducing end of the biopolymer. Thebiopolymer is first reacted with an organic disulfide compoundcontaining a terminal group, such as an amino group or a hydroxyl group,which is reactive with the terminal carboxyl group of the biopolymer.The reaction of the biopolymer and organic disulfide compound occursover a wide pH range, typically at a pH of from about 2.0 to 9.0.

In one aspect, the reaction of the biopolymer and therapeutic agentresults in the attachment of the biopolymer to the therapeutic agentthrough a disulfide bond. The linking group or spacer, which can be alower alkyl, separates the biopolymer from the therapeutic agent. Thelinking or spacer is a residue resulting from the cleavage of theorganic disulfide compound by the reactive thiol of the therapeuticagent.

Typical biopolymers include any of the polyanionic polysaccharides, suchas hyaluronic acid and any of its hyaluronate salts, such as sodiumhyaluronate, potassium hyaluronate, magnesium hyaluronate and calciumhyaluronate, carboxymethyl cellulose, carboxymethyl amylose,chondroitin-6-sulfate, dermatin sulfate, heparin, and heparin sulfate,as well as polyacrylic acid, polycarbophil, carboxymethyl chitosan,poly-α-glutamic acid, poly-γ-glutamic acid, carrageenan, and sodiumalginate. The common feature of the biopolymers of this invention isthat they are biocompatible, as that term is defined herein, theycontain carboxylic acid functionality, and they can be modified to reactwith an organic disulfide compound. Such modification can occur, forinstance, by reaction of the biopolymer with a suitable activatingagent, such as a carbodiimide, to render the carboxylic group vulnerableto nucleophilic attack by, for instance, an amine or a hydroxyl.Alternatively, the modification can occur at the terminal or end groupof the biopolymer by reduction of a terminal carbonyl group using aSchiff base.

In a preferred embodiment, the biopolymer is hyaluronic acid having amolecular weight in the range of from about 7.5×10² daltons to about1×10⁷ daltons. The hyaluronic acid is preferably activated by reactionwith an activating agent to render it vulnerable to nucleophilic attack.Suitable activating agents for this purpose include carbodiimides, suchas 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide.

The organic disulfide compound can be virtually any organic compoundhaving a disulfide bond. Preferably, the disulfide bond is positioned atone end of an alkyl chain, while the other end of the chain terminatesin a group reactive with the carbonyl group of the biopolymer.Preferably, the group that reacts with the biopolymer is an amino,carboxyl or hydroxyl group, but most preferably an amino group. Inaddition to being capable of reacting with the biopolymer, the organicdisulfide compound is also capable of reacting with the active thiolgroup of the therapeutic agent. Preferred organic disulfide compoundsinclude, in general, the nitro-pyridines, thio-pyridines, substitutedS-phenyl disulphides, S-sulfonate derivatives, 9-anthrymethylthioesters, S-carboxymethyl derivatives and nitro-thiobenzoic acidderivatives. More preferably, the organic disulfide compound is athio-nitro-pyridine, and most preferably3-nitro-2-pyridinesulfenyl-ethylamine.

The therapeutic agent is preferably one or more of the following: smallorganic molecules, proteins, nucleic acids, antibodies, peptides, aminoacids, lipids, polysaccharides, cell growth factors, and enzymes. Morepreferably, the therapeutic agent is native or recombinantcolony-stimulating factor (“CSF”), an amino acid or glucocerebrosidase.The therapeutic agent should contain a reactive thiol group to reactwith the modified biopolymer. The reactive thiol group can either beinherently part of the therapeutic agent, as in the case of cysteine, orthe reactive thiol group can be introduced into the therapeutic moleculeusing known techniques. For example, a free thiol group can beintroduced into a recombinant therapeutic protein molecule forconjugation and modification. Furthermore, some therapeutic drugs, suchas Captopril—a drug used to treat hypertension—inherently contain a freesulfhydryl group as shown in the structure below:

The amino groups of therapeutic agents can be conveniently convertedinto thiols by reaction with Traut's Reagent (aminothiolane).

The therapeutic agent is selected for the particular indication that isto be treated, and the biopolymer is selected, both as to its type andmolecular weight, for its ability to target a particular organ, cell ortissue. For instance, a therapeutic agent for treating Gaucher'sDisease, a serious liver ailment, is the enzyme glucocerebrosidase.Glucocerebrosidase can be targeted to the liver by forming a conjugatewith an appropriately sized hyaluronic acid molecule.

The biologically active conjugate of the present invention provides forimproved stability of the therapeutic agent as compared to the use ofthe unconjugated or unmodified therapeutic agent, or the use of othercarriers or conjugated compounds, such as polyethylene glycol (“PEG”) orlipids. The improved stability results in increased residence time inthe body of a subject and increased circulation time in the bloodstream. The conjugates of this invention also display improved targetingto specific tissues, organs and cells. Improved targeting is achievedthrough the selection of specific types and molecular weights of thebiopolymers.

In a further aspect, the invention involves the attachment of abiopolymer onto the surface of a substrate by means of a disulfidelinkage. The substrate can be a polymeric material, a ceramic or ametal. Preferably, the substrate is part of a medical device orinstrument, such as a stent, graft, suture, catheter, tubing orguidewire. The substrate is modified to contain an amino group, whichcan then be converted into a thiol group. The substrate can then bereacted with the biopolymer modified with the organic disulfide compoundto immobilize the biopolymer onto the substrate.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any method andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein,including published patent applications, and issued or granted patents,are hereby incorporated by reference in their entireties. Unlessmentioned otherwise, the techniques employed or contemplated herein arestandard methodologies well known to one of ordinary skill in the art.The materials, methods and examples are illustrative only and notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the UV analysis of hyaluronic acidmodified with 3-nitro-2-pyridinesulfenyl-ethylamine (Native andReduced). For Native HA/NEA, Absorbancy=1.1070 at 349 nm.

FIG. 2 is an H¹ NMR trace of hyaluronic acid modified with3-nitro-2-pyridinesulfenyl-ethylamine.

FIG. 3 is a trace of an IR spectra of hyaluronic acid modified with3-nitro-2-pyridinesulfenyl-ethylamine.

DETAILED DESCRIPTION OF THE INVENTION

The biologically active biopolymer-therapeutic agent conjugates of thepresent invention can be prepared by using a variety of chemicalpreparatory methods. An important feature of the conjugates of thisinvention is that the linkage between the therapeutic agent andbiopolymer contains a disulfide bond. The disulfide bond is formed bythe reaction of the therapeutic agent containing an active thiol withthe biopolymer, which has also been modified to contain a disulfidegroup by reaction with an organic disulfide compound. The procedure forpreparing the biopolymer-therapeutic agent conjugates of this inventionis described in more detail below.

Prior to the preparation of the conjugate, it is necessary to firstselect an appropriate biopolymer, and to modify the biopolymer so thatit can react with the therapeutic agent and form a disulfide bond. Thebiopolymer is selected from biocompatible polymers that contain acarbonyl group. The term “biocompatible”, as used herein, is intended todenote a substance that has no medically unacceptable toxic or injuriouseffects on biological function, or which is tolerated by the body.Examples of acceptable biopolymers include the polyanionicpolysaccharides, such as hyaluronic acid and any of its hyaluronatesalts, such as sodium hyaluronate, potassium hyaluronate, magnesiumhyaluronate and calcium hyaluronate, carboxymethyl cellulose (“CMC”),carboxymethyl amylose, carboxymethyl chitosan, chondroitin-6-sulfate,dermatin sulfate, heparin, and heparin sulfate, as well aspoly-α-glutamic acid, poly-γ-glutamic acid, carrageenan, and sodiumalginate. The term “polyanionic polysaccharide”, as used herein, isintended to mean polysaccharides containing more than one negativelycharged group, e.g. carboxyl groups at pH values above about a pH of4.0.

Biopolymers suitable for a particular application are selected from thisgroup of candidate biopolymers on the basis of their ability to targetparticular tissues, organs or cells, and their in vivo stability, i.e.the in vivo residence time in the circulatory system, or specifictissues, cells or organs. In a preferred embodiment, the biopolymer ishyaluronic acid having a molecular weight in the range of from about7.5×10² daltons to about 1×10⁷ daltons.

These biopolymers can be “activated” by reacting the biopolymer with asuitable activating agent to render the carboxylic group on thebiopolymer vulnerable to nucleophilic attack. Suitable activating agentsinclude carbodiimides, and preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidemethiodide. The reaction between the biopolymer and activating agentoccurs in an aqueous medium, preferably at a pH of from about 2.0 toabout 8.0, and more preferably a pH of from about 4.0 to about 5.1.Activation of the biopolymer can be useful if the therapeutic agent islinked to the intermediate carboxylic acid groups of the biopolymer.

The activated biopolymer is reacted with an organic disulfide compound.Suitable organic disulfide compounds can be selected from a wide rangeof molecules, including the nitro-pyridines, thio-pyridines, substitutedS-phenyl disulfides, S-sulfonate derivatives, 9-anthrymethyl thioesters,S-carboxymethyl derivatives and nitro-thiobenzoic acid derivatives, andpreferably the thio-nitro-pyridines. A particularly preferred organicdisulfide compound is 3-nitro-2-pyridinesulfenyl-ethylamine.

In one embodiment, the organic disulfide compound is a compound ofgeneral formulaR-L-S—S-Mwhere R is an amino, hydroxyl or carbonyl group, L, if present, is aspacer, preferably a lower normal or iso-substituted alkyl group, andmore preferably an ethyl group, each S is a sulfur atom, and M is anorganic moiety. The spacer, L, contains a terminal group that isreactive with the activated biopolymer. Preferably, the terminal groupis an amino, carboxyl or hydroxyl group, but most preferably an aminogroup. In addition to being capable of reacting with the biopolymer, theorganic disulfide compound is also capable of reacting with the activethiol group of the therapeutic agent.

The preparation of the preferred organic disulfide compound of thepresent invention, 3-nitro-2-pyridinesulfenyl-ethylamine, can beillustrated as follows:

As shown above, benzyl-3-nitro-2-pyridyl-sulfide is reacted withdichloroethane and sulfuryl chloride to prepare3-nitro-2-pyridinesulfenyl chloride. The 3-nitro-2-pyridinesulfenylchloride is reacted with 2-aminoethanethiol and formic acid to prepare3-nitro-2-pyridinesulfenyl-ethylamine as a precipitated product.

The activated biopolymer can then be reacted with the organic disulfidecompound as shown in the following reaction scheme:G-COOH+R-L-S—S-M→G-COR-L-S—S-Mwhere G is a biopolymer with a pendant carboxyl group, R is preferablyan amino group, L, if present, is a spacer, preferably a lower alkylgroup, each S is a sulfur atom, and M is an organic moiety. Preferably,the organic disulfide compound is 3-nitro-2-pyridinesulfenyl-ethylamine(“NEA”), and the reaction of NEA and hyaluronic acid, the preferredbiopolymer, can be illustrated as shown below, where “EDC” designates1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and “HOBt” designateshydroxybenzotriazole:

Alternatively, the biopolymer can be reacted with the organic disulfidecompound as shown in the following reaction scheme:G-CHO+R-L-S—S-M→G-C—R-L-S—S-Mwhere G, R, L S and M are as defined above. Preferably, the organicdisulfide compound is 3-nitro-2-pyridinesulfenyl-ethylamine (“NEA”), andthe reaction of NEA and hyaluronic acid, the preferred biopolymer, canbe illustrated as shown below, where NaCNBH₃ is sodium cyanoborohydride:

In the reaction scheme illustrated above, the biopolymer terminal ringopens as a result of a mutarotation equilibrium which occurs naturallyin carbohydrates. This forms a terminal aldehyde group, which is theonly aldehyde group in the molecule and can form a Schiff base. Thealdehyde reacts with the terminal amino group of the organic disulfidecompound. The addition of the sodium cyanoborohydride is a well knownreaction to reduce the resulting Schiff base. Other reagents which areknown to be able to reduce Schiff bases include sodium borohydride,lithium borohydride, lithium cyanoborohydride, sodium aluminum hydride,lithium aluminum hydride, tetrabutyl ammonium cyanobororhydride, sodiumamalgam, potassium graphite, and catalytic hydrogenation over platinumor nickel.

As illustrated above, this embodiment results in the attachment of theorganic disulfide compound to the reducing end of the biopolymer. Thispermits the reaction of one mole of organic disulfide compound per moleof biopolymer in a quantitatively controlled manner, which can be resultin higher yields, and more precise drug targeting and delivery.

The attachment of the organic disulfide compound need not be restrictedto aldehydes inherent in the biopolymer. One could introduce an aldehydeto the biopolymer by a reduction/oxidation sequence as described, forexample, by Raja, et al., Analytical Biochemistry 139: 168-177, 171(1984). Alternatively, one could attach an aldehyde to the biopolymer bymodifying an existing functional group of the biopolymer, such as ahydroxyl or carboxyl group. Methods for accomplishing this are wellknown in the chemical arts. Once the aldehyde is introduced or attachedto the biopolymer, the organic disulfide compound may be reacted withthe biopolymer as described herein.

The biopolymer-organic disulfide complex is then reacted with atherapeutic agent of choice. The therapeutic agent is selected based onthe particular disease state to be treated, and the organ, tissue orcell to be targeted. Suitable therapeutic agents include small organicmolecules, proteins, nucleic acids, antibodies, peptides, amino acids,lipids, polysaccharides, cell growth factors, and enzymes. Morepreferably, the therapeutic agent is native or recombinant colonystimulating factor, an amino acid or glucocerebrosidase.

Glucocerebrosidase is an enzyme which is used to treat a liver conditionknown as Gaucher's Disease. When glucocerebrosidase is selected as thetherapeutic agent, it is advantageous to also select hyaluronic acid,having an appropriate molecular weight, to target the therapeutic agentto liver cells.

The reaction of the therapeutic agent and the HA-NEA complex can beillustrated as shown below:

As shown in the above reaction scheme, the therapeutic agent of choicecontains an active thiol (—SH) group, that reacts with the HA-NEAconjugate, displacing the thio-nitro-pyridine residue. The therapeuticagent (shown above as the solid circle) is attached to the hyaluronicacid by a disulfide bond and an amine-terminated ethyl chain (spacer).The reaction occurs at a neutral to basic pH in the range of from about6-10.

The biologically active conjugates of this invention can be formulatedas pharmaceutical compositions for medical diagnosis or treatment,together with appropriate pharmaceutically acceptable carriers and,optionally, other therapeutic or diagnostic agents, using well knownformulation protocols. Administration of the pharmaceutical compositioncan be accomplished using an appropriate vehicle, such as tablets,implants, injectable solutions, and the like. Acceptable carriersinclude buffering agents and adjuvants. The precise amount of thebiologically active conjugate used in the pharmaceutical composition canbe determined based on the nature of the condition to be treated, andthe potency of the therapeutic agent used. This invention contemplatesboth local administration and time release modes of administration. Asused in this application, the term “subject” is intended to denote ahuman or non-human mammal, including, but not limited to, a dog, cat,horse, cow, pig, sheep, goat, chicken, primate, rat and mouse.

The process of the present invention can also be employed to modify thesurface of a medical device or instrument. A biopolymer, such ashyaluronic acid, can be immobilized onto the surface of a substratewhich has been modified to contain, for instance, exposed amino groups,which can be reacted with Traut's reagent and then HA-NEA as shownbelow:

The aminated surface, prepared, for instance, by cold plasma depositionof an allyl amine, is treated with a reagent, such as Traut's reagent,to convert the amino groups into free thiol groups. The derivatizedsurface is then reacted with HA-NEA to immobilize HA to the surface by adi sulfide bond. The advantage of this approach is the specificity ofthe reaction for the free sulfhydryl group between the surface and theactivated disulfide in the biopolymer. Under these reaction conditions,the activated biopolymer can only react with the surface and not withother biopolymer molecules, thereby creating a modified surface having awell defined biopolymer thickness. By contrast, the use of exogenouslyadded activating agents, such as glutaraldehyde and carbodiimide, toachieve similar results, can result in interpolymer covalent bondformation that can cause uncontrolled increases in biopolymer coatingthicknesses. Another advantage is the use of mild reaction conditions,such as the use of an aqueous solvent, ambient temperatures, and a pH inthe range of from about 6-10.

This surface modification approach can be used to modify the surfacecharacteristics of stents, to prevent platelet activation andaggregation, or catheter surfaces, to inhibit cell adhesion. Anadditional advantage of this approach is that the HA will only reactwith the surface, and not with itself, so the thickness and compositionof the HA layer can be readily controlled.

From the above description, one skilled in the art can readily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope of thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

As one skilled in the art will appreciate, particular details of themethods of this invention may differ from certain of those methodsexemplified herein.

The following examples of the invention are provided by way ofillustration only, and are not intended to limit the invention set forthin the appended claims.

EXAMPLE 1 Synthesis of 3-nitro-2-pyridinesulfenyl-ethylamine (NEA)

Benzyl-3-nitro-2-pyridyl sulfide (10 grams, 40.6 mmol.), that had beenazeotropically dried by coevaporation with toluene, was dissolved in1,1-dichloroethane (21 mL). The reaction solution was cooled to 0° C.,and sulfuryl chloride (4.24 mL, 52.78 mmol) was added, followed bytriethylamine (100 μL, 1.4 mmol). This resulted in a precipitate thatwas collected, washed with hexane, dried under reduced pressure, and wasused without further purification.

The 3-nitro-2-pyridinesulfenyl chloride was added to a solution of2-aminoethanethiol (4.1 grams, 36.19 mmol) in 230 mL of 90% formic acid.The solution was vigorously stirred for one hour. The resultingprecipitate was removed by filtration, and a large volume ofdiethylether was added to the supernatant. The precipitate wascollected, dissolved in warm methanol, and reprecipitated with diethylether. 3-Nitro-2-pyridinesulfenyl-ethylamine was obtained in 68% overallyield.

EXAMPLE 2 Synthesis of HA-NEA with 1%-2% NEA Modification

1-Hydroxybenzotriazole (“HOBt”) (16.8 mg, 124 μmol) was added to an 8.0%solution (312 μL, 62 μmol) of 70 kdalton HA. To this solution was addedNEA (25.0 mg, 93 μmol). The pH of the reaction mixture was adjusted to3.0 by the addition of 2M HCl.1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (“EDC”) (35.7 mg, 186μmol) was added to the acidified reaction mixture, and the mixture wasstirred at room temperature for 1.5 hours. All of the above reagentswere dissolved in sufficient water to achieve a final HA concentrationin the reaction solution of 1%. The reaction was purified by dialysisagainst phosphate buffered saline (“PBS”) for 24 hours, followed bydialysis against water for an additional 24 hours. The product wasrecovered by lyophilization.

EXAMPLE 3 Synthesis of HA-NEA with 15%-20% NEA Modification

HOBt (16.8 mg (124 μmol) was added to an 8.0% solution (312 μL, 62 μmol)of 70 kdalton HA, followed by the addition of NEA (25.0 mg, 93 μmol).The pH of the reaction mixture was adjusted to 3.0 by the addition of 2MHCl. EDC (71.3 mg, 372 μmol) was added to the acidified reactionmixture, and the mixture was stirred at room temperature for 1.5 hours.All of the above reagents were dissolved in sufficient water to achievea final HA concentration in the reaction solution of 1%. The reactionwas purified by dialysis against phosphate buffered saline (“PBS”) for24 hours, followed by dialysis against water for an additional 24 hours.The product was recovered by lyophilization.

EXAMPLE 4 Synthesis of HA-NEA with 40%-50% NEA Modification

HOBt (16.8 mg, 124 μmol) was added to an 8.0% solution (312 μL, 62 μmol)of 70 kdalton HA, followed by the addition of NEA (25.0 mg, 93 μmol).The pH of the reaction mixture was adjusted to 3.0 by the addition of 2MHCl. EDC (178.3 mg, 930.1 μmol) was added to the acidified reactionmixture, and the mixture was stirred at room temperature for 1.5 hours.All of the above reagents were dissolved in sufficient water to achievea final HA concentration in the reaction solution of 1%. The reactionwas purified by dialysis against phosphate buffered saline (“PBS”) for24 hours, followed by dialysis against water for an additional 24 hours.The product was recovered by lyophilization.

EXAMPLE 5 Synthesis of HA-NEA Using a Mixed Solvent Mixture of 50 PartsN-methylpyrrolidone (“NMP”) and 50 Parts Water

HOBt (16.8 mg, 124 μmol) was added to an 8.0% solution (312 μL, 62 μmol)of 70 kdalton HA, followed by the addition of NEA (25.0 mg, 93 μmol).The pH of the reaction mixture was adjusted to 3.0 by the addition of 2MHCl. EDC (35.7 mg, 186 μmol) was added to the acidified reactionmixture, and the mixture was stirred at room temperature for 1.5 hours.All of the above reagents were dissolved in a sufficient amount of a50:50 NMP/water mixture to achieve a final HA concentration in thereaction solution of 1%. The reaction was purified by dialysis againstphosphate buffered saline (“PBS”) for 24 hours, followed by dialysisagainst water for an additional 24 hours. The product was recovered bylyophilization.

EXAMPLE 6 Synthesis of HA-NEA Using a Mixed Solvent Mixture of 50 PartsEthyl Alcohol and 50 Parts Water

HOBt (16.8 mg, 124 μmol) was added to an 8.0% solution (312 μL, 62 μmol)of 70 kdalton HA, followed by the addition of NEA (25.0 mg, 93 μmol).The pH of the reaction mixture was adjusted to 3.0 by the addition of 2MHCl. EDC (35.7 mg, 186 μmol) was added to the acidified reactionmixture, and the mixture was stirred at room temperature for 1.5 hours.All of the above reagents were dissolved in a sufficient amount of a50:50 EtOH/water mixture to achieve a final HA concentration in thereaction solution of 1%. The reaction was purified by dialysis againstphosphate buffered saline (“PBS”) for 24 hours, followed by dialysisagainst water for an additional 24 hours. The product was recovered bylyophilization.

EXAMPLE 7 Synthesis of HA-NEA Using a High Molecular Weight HA

HOBt (33.5 mg, 248 μmol) was added to a 1.0% solution (5 mL, 124 μmol)of 100 kdalton HA, followed by the addition of 50.0 mg (186 μmol) ofNEA. The pH of the reaction mixture was adjusted to 3.0 by the additionof 2M HCl. EDC (142.7 mg, 744 μmol) was added to the acidified reactionmixture, and the mixture was stirred at room temperature for 1.5 hours.All of the above reagents were dissolved in sufficient water to achievea final HA concentration in the reaction solution of 0.2%. The reactionwas purified by dialysis against phosphate buffered saline (“PBS”) for12 hours, followed by dialysis against water for an additional 12 hours.The product was recovered by lyophilization.

EXAMPLE 8 HA-NEA Characterization

UV analysis of the HA-NEA complex, prepared as described above, revealsa λ max at 345 nm, corresponding to the 3-nitro-2-pyridinesulfenyl groupwhich results in a bathochromic shift to 401 nm upon reduction with DTT(FIG. 1). The ¹H NMR shows a distinct set of peaks in the aromaticregion generated from the 3-nitro-2-pyridinesulfenyl group (FIG. 2).Evidence of the modified HA structure can also be found in the IRspectra (FIG. 3). An amide stretch is observed at 1655 cm⁻¹, while theHA carboxyl peak at 1655 cm⁻¹ is reduced. The IR spectrum containsadditional peaks that can be attributed to the3-nitro-2-pyridinesulfenyl group, specifically, the aromatic nitro andpyrindinyl stretches occurring at 1557 cm⁻¹ and 746 cm⁻¹, respectively.

EXAMPLE 9 Synthesis of End-Linked HA-NEA with 35% NEA Modification

NEA (64.5 mg, 240 μmol) was added to 10 mL of a 2.0% solution of HA (60kdalton, pH 4.1). The reaction was stirred at room temperature for 24hours, at which time 151 mg (2.4 mmol) of sodium cyanoborohydride wasadded. The mixture was then stirred for one hour. The mixture wasfiltered through a 0.45 μm filter and purified by dialysis against 1MNaCl for 24 hours, followed by dialysis against PBS and water for anadditional 48 hours. The product was recovered by lyophilization.

EXAMPLE 10 Synthesis of End-Linked HA-NEA with 50% NEA Modification

NEA (64.5 mg, 240 μmol) was added to 10 mL of a 10.0% solution of HA (60kdalton, pH 4.1). The reaction was stirred at room temperature for 24hours, at which time 151 mg (2.4 mmol) of sodium cyanoborohydride wasadded. The mixture was then stirred for one hour. The mixture wasfiltered through a 0.45 μm filter and purified by dialysis against 1MNaCl for 24 hours, followed by dialysis against PBS and water for anadditional 48 hours. The product was recovered by lyophilization.

EXAMPLE 11 Attachment of Cysteine to HA

Dansyl-L-cysteine, in the amount of 2.25 equivalents relative to theamount of 3-nitro-2-pyridinesulfenyl, was added to an HA-NEA (4 mg, 10μmol) complex. The pH was adjusted to 6.5 with 0.5 M HCl, and thereaction was dialyzed against PBS for 12 hours, followed by dialysisagainst water for an additional 12 hours. The retentate was thenlyophilized to form an HA-cysteine conjugate with the quantitativeincorporation of cysteine relative to the amount of3-nitro-2-pyridinesulfenyl modification.

EXAMPLE 12 Attachment of Cysteine to HA

Dansyl-L-cysteine, in the amount of 2.25 equivalents relative to theamount of 3-nitro-2-pyridinesulfenyl, was added to of an HA-NEA (4 mg,10 μmol) complex. The pH was adjusted to 8.0 with 0.5 M HCl, and thereaction was stirred at room temperature for 2 hours. The reactionmixture was dialyzed against PBS for 12 hours, followed by dialysisagainst water for an additional 12 hours. The retentate was thenlyophilized to form an HA-cysteine conjugate with the quantitativeincorporation of cysteine relative to the amount of3-nitro-2-pyridinesulfenyl modification.

EXAMPLE 13 Attachment of Cysteine to HA

Dansyl-L-cysteine, in the amount of 2.25 equivalents, relative to theamount of 3-nitro-2-pyridinesulfenyl, was added to an HA-NEA (4 mg, 10μmol) of complex. The pH was adjusted to 10.0 with 0.5 M HCl, and thereaction was stirred at room temperature for 2 hours. The reactionmixture was dialyzed against PBS for 12 hours, followed by dialysisagainst D.I. water for an additional 12 hours. The retentate was thenlyophilized to form an HA-cysteine conjugate with the quantitativeincorporation of cysteine relative to the amount of3-nitro-2-pyridinesulfenyl modification.

EXAMPLE 14 Attachment of Cysteine to the Reducing-End of HA

Dansyl-L-cysteine, in the amount of 2.25 equivalents, relative to theamount of 3-nitro-2-pyridinesulfenyl, was added to 43 mg (0.64 umol) ofan HA-NEA complex. The reaction was stirred at room temperature for 2hours. The reaction mixture was dialyzed against 0.1 M NaCl for 12hours, followed by dialysis against D.I. water for an additional 12hours. The retentate was then lyophilized to form a HA-Cysteineconjugate.

EXAMPLE 15 Attachment of Ribonuclease A to HA

Dansyl-L-cysteine, in the amount of 2.25 equivalents, relative to theamount of 3-nitro-2-pyridinesulfenyl, was added to an HA-NEA (4 mg, 10μmol) complex. The pH was adjusted to 10.0 with 0.5 M HCl, and thereaction was stirred at room temperature for 2 hours. The reactionmixture was dialyzed against PBS for 12 hours, followed by dialysisagainst D.I. water for an additional 12 hours. The retentate was thenlyophilized to form an HA-cysteine conjugate with the quantitativeincorporation of cysteine relative to the amount of3-nitro-2-pyridinesulfenyl modification.

Although the foregoing invention has been described in terms of certainpreferred embodiments, other modifications, variations and changes canreadily be made as will be appreciated by those skilled in the art. Itis to be understood that all such modifications, variations and changesare intended to fall within the scope of the present invention asdefined by the appended claims.

1. A biologically active conjugate of a biopolymer and a therapeuticagent comprising a compound of formula; or a pharmaceutically acceptablesalt thereof:G-C(O)—R-L-SS—B wherein G-C(O)— is a biopolymer comprising at least onecarbonyl group, —C(O)—, on the biopolymer backbone bound to R, and R isan amino group or an oxygen atom, orG-C—R-L-SS—B Wherein G-C— is a biopolymer having a methylene group, C,bound to R, and R is an imino or amino group; and wherein L is loweralkyl spacer, B is a therapeutic agent, and each S is a sulfur atom. 2.The conjugate of claim 1, wherein the spacer is a lower normal oriso-substituted alkyl group.
 3. The conjugate of claim 2, wherein thespacer is an ethyl group.
 4. The conjugate of claim 1, wherein thebiopolymer is selected from the group consisting of hyaluronic acid,carboxymethyl cellulose, carboxymethyl amylose, carboxymethyl chitosan,chondroitin-6-sulfate, dermatin sulfate, polycarbophil, heparin, andheparin sulfate.
 5. The conjugate of claim 4, wherein the biopolymer ishyaluronic acid.
 6. The conjugate of claim 5, wherein the hyaluronicacid has a molecular weight in the range of from about 7.5×10² daltonsto about 1×10⁷ daltons.
 7. The conjugate of claim 1, wherein thebiopolymer is selected from the group consisting of polyacrylic acid,poly-α-glutamic acid, poly-γ-glutamic acid, carrageenan, calciumalginate and sodium alginate.
 8. The conjugate of claim 1, wherein thetherapeutic agent is selected from the group consisting of small organicmolecules, proteins, peptides, nucleic acids, antibodies, amino acids,lipids, polysaccharides, cell growth factors, and enzymes.
 9. Theconjugate of claim 8, wherein the therapeutic agent is a native orrecombinant colony stimulating factor.
 10. The conjugate of claim 8,wherein the therapeutic agent is an amino acid.
 11. The conjugate ofclaim 8, wherein the therapeutic agent is glucocerebrosidase.
 12. Theconjugate of claim 1, wherein the conjugate is of the formulaG-C(O)—R-L-SS—B.
 13. The conjugate of claim 1, wherein the conjugate isof the formula G-R-L-SS—B.
 14. A pharmaceutical composition comprisingthe conjugate of claim 1, and a pharmaceutically acceptable carrier. 15.The pharmaceutical composition of claim 14, wherein the biopolymer isselected to target cancer cells.
 16. The pharmaceutical composition ofclaim 14, wherein the biopolymer is selected to target liver cells. 17.The pharmaceutical composition of claim 14, wherein the biopolymer isselected to target spleen cells.
 18. The pharmaceutical composition ofclaim 14, wherein the therapeutic agent is native or recombinant colonystimulating factor.
 19. The pharmaceutical composition of claim 14,which is formulated to provide sustained in vivo release of thebiologically active conjugate.
 20. A method for treating a subjectcomprising administering to the subject the pharmaceutical compositionof claim
 14. 21. The method of claim 20, wherein the pharmaceuticalcomposition has enhanced in vivo stability in a subject.
 22. The methodof claim 20, wherein the pharmaceutical composition targets liver cells.23. The method of claim 20, wherein the pharmaceutical compositiontargets cancer cells.